Abstract Elucidation of the molecular mechanisms that underlie disease is crucial for the development of new therapeutic agents. Researchers have recently developed a number of methods to identify the genes, proteins, and metabolites associated with disease. However, complementary methods that define connections between these molecules[unreadable]connections that are the foundation of biological models of disease and targeted medicine[unreadable]have proven much more difficult to develop. As a result, there remains a tremendous need for innovative new approaches that reveal interactions between the molecular components of disease in vivo. The following proposal outlines the continued development and application of one such method, termed discovery metabolite profiling (DMP), for the assignment of endogenous substrates to the prolyl peptidase family of enzymes. DMP integrates an array of biological and chemical methods, including genetics, pharmacology, and analytical chemistry to identify bona fide physiological enzyme-substrate interactions. Importantly, by using DMP to study a family of enzymes that are virtually lacking in known endogenous substrates, but regulate phenotypes of tremendous biomedical interest, this research will begin to realize the incredible potential of the prolyl peptidases in medicine. Furthermore, the application of DMP to peptidases will demonstrate the generality of this approach for the future characterization of medically relevant enzymes and signaling pathways.